蛋白激酶 CK2 是一種具有多種功能的絲胺酸/蘇胺酸蛋白質激酶，CK2 作用的受質眾多且廣泛表現在哺乳類動物細胞中，對於細胞週期的發展、轉錄作用以及抗細胞凋亡等機制扮演非常重要的角色。在神經系統中，CK2 已知可以保護神經細胞以抵抗外來的傷害，但是其分子層面的機制目前尚未釐清。本篇論文的研究重點在於探討 CK2 保護作用可能參與的細胞分子機制。血清反應因子 SRF 是一種哺乳類動物細胞的轉錄因子，調控基因的轉錄作用來促進細胞的存活。Mcl-1 是抗細胞凋亡家族 Bcl-xL 家族蛋白成員之一，可以促進細胞的存活能力。先前研究指出，SRF 會受到 CK2 的磷酸化作用而增加本身的 DNA 結合能力。在其他研究也指出，Mcl-1 會受到 SRF 的調控。在本篇論文的第一部份，著重於 Mcl-1 的表現是否會受到 CK2 調控 SRF 的路徑所影響，實驗結果顯示，轉染野生型 CK2α 質體 DNA 可以增加海馬迴 CA1 腦區的 SRF 磷酸化，而轉染不活化的突變型 CK2αΑ156 質體 DNA 則會減少 SRF 的磷酸化。更進一步，轉染野生型 CK2α 會增加 Mcl-1 的 mRNA 及蛋白質表現，轉染突變型 CK2αΑ156 則減少 Mcl-1 的表現。此外，轉染突變型 SRF99A 也會減少 Mcl-1 的 mRNA 及蛋白質表現；而且在共同轉染實驗中，SRF99A 會拮抗野生型 CK2α 對促進的 Mcl-1 蛋白質表現的作用。
另一方面，DARPP-32 是一個在新紋狀體神經細胞中具有調控多巴胺訊息效力的訊息傳遞分子。先前研究指出，DARPP-32 具有抗細胞凋亡的功能，且發現在 DARPP-32 Ser102 氨基酸會受 CK2 的磷酸化作用。因此，本篇論文的第二部份主要是探討 CK2 的抗細胞凋亡能力是否是透過磷酸化 DARPP-32 來調控。實驗結果顯示，轉染野生型 CK2α 可以增加紋狀體 DARPP-32 的磷酸化，而轉染不活化的突變型 CK2αΑ156 則會減少 DARPP-32 的磷酸化。此外，轉染 CK2α 的小干擾 RNA (siRNA) 可以抑制內生性的 CK2 表現，同時也會減少 DARPP-32 的磷酸化以及抗細胞凋亡蛋白, Bcl-xL 的表現。綜合這些實驗結果，CK2α可以分別透過 SRF 或 DARPP-32 調控的訊息傳遞來促進 Mcl-1 或 Bcl-xL 的表現進而調控神經系統的抗細胞凋亡機制。 / Protein kinase CK2 is a multifunctional serine/threonine protein kinase with many protein substrares and is ubiquitously expressed in mammalian cells to play an important role in cell cycle progression, transcription, and anti-apoptosis. In the nervous system, CK2 is shown to protect neurons against injury, but the cellular mechanisms are not well studies. In the present studies, we investigate which cellular mechanism might involve in the CK2 protection effects. The serum response factor (SRF) is a mammalian transcription factor which mediates some gene transcriptions relevent to promote the cell survival. The Myeloid cell leukemin 1 (Mcl-1) is one of the anti-apoptotic Bcl-2 family members and is involved in promoting cell viability. Previous studied have revealed that the SRF phosphorylation by CK2 can enhance its DNA-binding activity. The regulation of Mcl-1 by SRF has also been reported in other studies. In the first part of the present studies, we investigate whether the Mcl-1 expression is regulated by CK2 through SRF mediated pathway. The results from wildtype CK2α plasmid DNA transfection revealed that the phosphorylated SRF were increased in hippocampus CA1 region, whereas transfection of the catalytically inactive CK2αA156 mutant plasmid DNA decreased phosphorylated SRF. Further, wildtype CK2α increased, whereas CK2αA156 mutant decreased the mRNA and protein levels of Mcl-1. Moreover, transfection of the mutant SRF99A also decreased the mRNA and protein levels of Mcl-1. Furthermore, the mutant SRF99A antagonized the upregulatory effects of wildtype CK2α on Mcl-1 protein level in the co-transfection experiments.
In the other side, DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa) is a signal transduction molecule that regulates the efficacy of dopamine signaling in neostriatal neurons. Previous studies have revealed that DARPP-32 might involve in the anti-apoptosis and its Ser102 residue is phosphorylated by CK2. Therefore, in the second part of this study, we investigate whether one of the anti-apoptotic effects of CK2 is through DARPP-32 phosphorylation by CK2 in the present study. The results revealed that the phosphorylated DARPP-32 is increased in stratum by wildtype CK2α transfection and decreased by catalytically inactive CK2αA156 mutant transfection. Further, transfection of CK2α siRNA can inhibit endogenous CK2 expression and also decrease phosphorylation of DARPP-32 as well as the anti-apoptotic protein, Bcl-xL. These results together suggest that CK2α-mediated anti-apoptotic effects are partially through SRF mediated or DARPP-32 mediated signaling to regulate Mcl-1 or Bcl-xL expression, respectively.
| Identifer | oai:union.ndltd.org:CHENGCHI/G0096754002 |
| Creators | 張家銘 |
| Publisher | 國立政治大學 |
| Source Sets | National Chengchi University Libraries |
| Language | 中文 |
| Detected Language | English |
| Type | text |
| Rights | Copyright © nccu library on behalf of the copyright holders |
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